PROTEIN EXTRACTION USING Reverse Micelles (RMs)

Liquid-liquid extraction is a useful method to separate components (compounds) of a mixture.

The success of this method depends upon the difference in solubility of a compound in various solvents

Reversed Micelles Extraction

Surfactant form a kind of aggregate in non-polar organic solvent.

When the concentration of surfactant exceed the

critical micellar concentration, surfactant can form a kind of aggregate in water solution.
micelles and reversed micelles

Hydrophilic group

(Water loving)

Hydrophobic group

Air liquid interface

(Water hating) Aqueous phase

Amphiphilic molecule

Surfactants arranged at interface

O O H3C S O Molecular Formula = C12 H25 Na O3 S Na

Sodium dodecyl sulfate (SDS)

H3C

Na O O S O O

CH3

CH3

H3C

Molecular Formula = C32 H62 Na2 O10 S2

Sodium bis (2-ethylhexyl) sulphosuccinate (AOT)

CH3

Molecular Formula = C17 H30 N

Cetyl pyridinium bromide

O H3C O H Molecular Formula = C12 H26 O2

Polyoxyethylenes

Brij-30

Classification of surfactants
Classified by the hydrophilic group: Anionic Carboxylic Sulfuric esters Alkane sulfonic acids Alkyl aromatic sulfonic acids Others: Phosphates, phosphoric acids, Cationic Amine salts (primary, secondary, and tertiary) Quaternary salts

Nonionic
Ethers Esters Amides Amphoteric

 Class of aggregates formed from amphiphilic molecules (contains both polar and apolar group)

Surface tension

Critical micelle concentration (CMC)

Reduction in surface tension takes place

Conc(surfactant)

No change in surface tension after CMC

Micelles formed only above the CMC

P < 0.33 Spherical/ellipsoidal

P = 0.33-0.5 Cylindrical/rodlike

P alc
= Surfactant molecular volume lc = length of the hydrocarbon chain a = Area per polar head group of surfactant

P = 0.5-1.0 bilayers/vesicles

P > 1 Reverse/Inverted micelles

 nanometer scale (3-5nm)droplet of aqueous solution stabilized in an apolar environment by the presence of surfactant layer at the interface.
Aqueous core Oil phase

Aggregates form in such a way that the surfactant polar heads orient inwards encapsulating an aqueous micro-domain

Reverse micelles are also called w/o micro-emulsions.

Empty micelle

Filled micelle

Surfactants reduce surface tension when adsorbed at the interface at low concentration

Organic Phase

Surfactant molecules selfassociate to form aggregates

Protein

Aqueous phase

The concentration above which the aggregates are formed is called critical micelle concentration (CMC)

Mechanism of protein solubilization in reverse micelles

 Reverse micelles should be capable of selective solubilization of proteins, called forward extraction

Water

Oil

Also it should be able to release the purified protein back into the organic phase, called backward extraction.

Forward and backward extraction of proteins is governed by several parameters. Forward Extraction Backward Extraction

 Aqueous phase parameters

pH Ionic strength

Organic phase parameters

Type of surfactant/solvent Presence of co-surfactant

Protein characteristics
pI Size and Shape

Others
Temperature

Type of salt

Hydrophobicity/charge distribution

 Those which can form a reverse micelle (AOT, CTAB, TOMAC etc)

Anionic/Cationic ?
pH < pI
Protein positively charged

pH > pI
Protein negatively charged

Electrostatic interaction mainly responsible for protein extraction

Surfactant Concentration

Effect of Salt (Ionic strength)

Forward Extraction (FE)

Backward Extraction (BE)

Forward Extraction (FE)

Backward Extraction (BE)

Increasing favours FE

conc.

Increasing conc. does not favor BE

At higher conc. precipitation of proteins may also occur.

Lower salt concentration favorable (less debye screening effect)

Higher salt concentration favorable.

Optimum concentration need to be found out.

Optimum concentration need to be found out.

Reverse micellar extraction of Hen Egg Lysozyme Refer to this paper Work in groups 2 people per group

Discuss the following:

1. 2. 3. 4. What was the objective of this experiment ? What were the different parameters studied? Outline the extraction procedure What were the optimum results obtained and critically evaluate the results.